Multiple nucleotide variants in genetic diagnosis: implications from 11,467 cases of hearing loss.

Multiple nucleotide variants (MNVs) are frequently misannotated as separate single-nucleotide variants (SNVs) by widely utilized variant-calling pipelines, presenting substantial challenges in genetic testing and research. The role of MNVs in genetic diagnosis remains inadequately characterized, particularly within large disease cohorts. In this study, we comprehensively investigate codon-level MNVs (cMNVs) across 157 hearing loss (HL)-related genes in 11,467 HL cases and 7258 controls from the Chinese Deafness Gene Consortium (CDGC) cohort. A total of 116 cMNVs are identified, occurring in 29.07% of HL cases. Among them, 56.03% of cMNVs exhibit functional consequences distinct from constituent SNVs. Moreover, amino acid substitutions exclusive to cMNVs cause more substantial physicochemical disruptions than those associated with SNVs. Notably, 51 cMNVs show pathogenicity classifications that diverge from at least one constituent SNV, impacting genetic interpretation in 145 cases. Pathogenicity interpretation of cMNV facilitates definitive genetic diagnoses in eight HL cases that would otherwise have been subject to misdiagnoses or missed diagnoses. These findings provide critical insights into the genomic characteristics, functional impacts, and diagnostic implications of cMNVs, underscoring their clinical significance in genetic diagnosis and emphasizing the necessity for comprehensive and accurate detection and interpretation of cMNVs in genetic testing and research.